The term neurodegenerative disorder is used to describe diseases characterized by the progressive breakdown of neuronal function and structure. This term encompasses disorders such as Alzheimer's, Parkinson's, and Huntington's diseases, as well as amyotrophic lateral sclerosis (ALS), among others, although neuronal damage is also associated with stroke and ischemic events, cerebral palsy, and head trauma. Although the human and economic cost of neurodegeneration continues to be astronomical, treatment is largely limited to palliative care and prevention of symptom progression. Therefore, there is a constant demand for novel and effective approaches to slow or prevent the progression of these diseases.
One target under investigation is neuronal nitric oxide synthase (nNOS). Nitric oxide (NO) is an important second messenger in the human body, and dysregulation of its production is implicated in many pathologies. NO is produced by the nitric oxide synthase enzymes, of which there are three isoforms: endothelial nitric oxide synthase (eNOS), which regulates blood pressure and flow, inducible nitric oxide synthase (iNOS), involved in immune system activation, and nNOS, which is required for normal neuronal signaling. Nonetheless, over-expression of nNOS in neural tissue and increased levels of NO can result in protein nitration and oxidative damage to neurons, especially if peroxynitrite is formed from excess NO. Indeed, overexpression of nNOS or excess NO has been implicated in or associated with many neurodegenerative disorders. The inhibition of nNOS is, therefore, a viable therapeutic strategy for preventing or treating neuronal damage.
All NOS enzymes are active only as homodimers. Each monomer consists of both a reductase domain with FAD, FMN, and NADPH binding sites, and a heme-containing oxygenase domain, where the substrate (L-arginine) and cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H4B) bind. Activated and regulated by calmodulin binding, electron flow proceeds from one monomer's reductase domain to the other's oxygenase domain, catalyzing the oxidation of arginine to citrulline with concomitant production of NO. (See, Rosen, G. M.; Tsai, P.; and Pou, S. Mechanism of free-radical generation by nitric oxide synthase. Chem. Rev. 2002, 102 (4), 1191-1199.)
Not unexpectedly, most investigated nNOS inhibitors are mimetics of arginine and act as competitive inhibitors. One major challenge in designing nNOS inhibitors is that eNOS and iNOS share high sequence similarity and an identical overall architecture with nNOS, especially in their substrate-binding sites. Lack of isoform selectivity could have deleterious effects; inhibition of eNOS can cause severe hypertension, and iNOS inhibition could impair immune system activation. Previously, fragment hopping and subsequent structure-based optimization afforded compounds 1 and 2 (representative nNOS inhibitors are shown in FIG. 1). These compounds are highly potent and selective nNOS inhibitors, and compound 1 reverses a hypoxic-ischemic brain damage phenotype in newborn rabbit kits when administered intravenously to the dam. (See, Ji, H.; Tan, S.; Igarashi, J.; Li, H.; Derrick, M.; Martásek, P.; Roman, L. J.; Vasquez-Vivar, J.; Poulos, T. L.; and Silverman, R. B. Selective Neuronal Nitric Oxide Synthase Inhibitors and the Prevention of Cerebral Palsy. Ann. Neurol. 2009. 65, 209-217.)
Although effective, compounds 1 and 2 suffer from several drawbacks. Like most arginine mimics, they are very polar and hydrophilic and contain numerous basic moieties and hydrogen-bond donors, as well as many rotatable bonds and a high total polar surface area (tPSA), all properties that hamper both GI absorption and blood-brain barrier permeation. Many attempts to improve the bioavailability of these compounds have been made, including alkylation, fluorination, introduction of lipophilic tails, and replacement of amine moieties—most of these strategies either diminished potency or selectivity or were synthetically challenging. The chiral scaffolds of 1 and 2 are also difficult (>12 steps) to prepare, making them less desirable, from a clinical standpoint, than simpler scaffolds such as that of a compound 3 and commercial candidate 4; (potencies and selectivities given in FIG. 1). Nonetheless, these simplified molecules are not without fault; their isoform selectivities are lower, 3 suffers from poor Caco-2 permeability, and 4 is much less potent in cell-based assays than against isolated enzymes—both likely the result, in part, of the amidine moiety, which will be charged at physiological pH.
Accordingly, the design of NOS inhibitors remains an on-going concern in the art. In particular, the search continues for compounds providing good bioavailability without compromising potency and/or selectivity, while offering the advantages and benefits associated with ease of preparation and molecular variation.